This web page was produced as an assignment for an undergraduate immunology class at Davidson College.

RAG-1 and RAG-2


The Human Immune System functions due to the ability of B cells and T cells to recognize Antigens and create the necessary response to neutralize the pathogen. Antigens are recognized in the body through highly specific interactions with the variable immnoglobulin domains on B cells and the T cell receptor region of T cells. Through the process of somatic recombination a variety of specific immunoglobulin domains and T cell receptors can be created with minimal genetic requirements. The Recombination Activating proteins RAG -1 and RAG-2 act together as a heterodimer in the initiation of the process of antigen receptor gene segment assembly. (Spanopoulou, 1996). These proteins are encoded by the Recombination Activating genes, which are found on human chromosome 11 (Shigeoka, 2002). The RAG genes are in very close proximity and consist of one exon (Janeway, 2001). These proteins are essential for proper formation and function of B cell and T cell receptors. (Janeway, 2001)These proteins are active only in the very early stages of lymphoid development as part of the V(D)J recombinase. These enzymes act together to effect somatic recombination. The RAG complex is responsible for initiating the recombination of the variable (V), diversity (D) and joining (J) gene segments of the receptor (Janeway 2001).


The genetic recombination of the V, D, and J coding regions of B cell and T cell receptors is the only known form of site-specific DNA rearrangement in vertebrates.(Wenhui et al., 2001).This process is mediated by Recombination Signal Sequences (RSS). RSS sequences consist of a specified conserved block of seven (heptamer) or nine (nonamer) nucleotides preceding and trailing the desired genetic sequence. Recombination only occurs between gene segments on the same chromosome and must be flanked by an RSS and separated by spacers of either 12 or 23 base pairs. The 12/23 rule allows only segments flanked by 12 base pair spacers to be joined to 23 base pair segments. This ensures that the correct segments are connected to one another. This recombination allows for a great deal of genetic diversity among T cell and B cell receptors. The diverse range of receptors allow for different binding specificity and affinity for many different antigens (Bassing,2002).


The RAG proteins align the two RSS marked regions that are going to bind. More specifically, the RAG-1 protein is presumed to recognize the nonamer of the RSS. After this initial alignment the endonuclease of the RAG complex make two double stranded cuts on the 5' end of the RSS leaving a free 3' OH group. (Janeway, 2001). The OH group then acts to hydrolyze the phosphoester bond on the other end which creates a DNA hairpin of the gene segment coding region (Janeway, 2001). The DNA ends that are created as a result do not float apart because they are held tightly in complex by the RAG proteins until the join is completed (Janeway, 2001).


The stepwise role of the RAG protein complex in V(D)J recombination is as follows: (Taken from Janeway, 2001)

  1. The RAG protein complex binds to 12 and 23 base pair RSS.
  2. The RAG proteins then bind to each other which bridges together the segments to be joined
  3. The RAG complex then cleaves one strand of the double stranded DNA
  4. Once cleaved the 5' end of the cut end reacts with the complementary uncut strand.
  5. This causes a break which leaves and double-stranded break at the end forming a hairpin loop.
  6. The hairpin is cleaved at a random point and additional bases are added by deoxynucleotidyl transferase to generate imprecise ends.
  7. DNA ligase joins the ends of the gene segments to form the coding joint and the RSS ends which form the signal joint.

coding joint: The V and J segments remaining on the chromosome join together to form a coding joint

signal joint: The ends of the heptamer region of the DNA molecule joined in a head to head fashion forming a signal joint in a circular piece of extra chromosomal DNA which is later lost.

RAG-1 and RAG-2 are only active during the initial phases of the V(D)J recombination. In each recombination even there are two chances, one on each loci, for a successful recombination. After a recombination event has successfully competed at either loci the RAG complex is inactivated for the duration of the cell proliferation that follows (Janeway, 2001).


**Although RAG-1 and RAG-2 are both essential for initiation of V(D)J recombination, the actual DNA binding activity of RAG-2 is unknown. RAG-2 collaborates with RAG-1 in recognition of recombination signal sequences. In the absence of RAG-2 the binding of RAG-1 is weakened (Wenhui, 2001).


"Mutations in the RAG genes result in severe combined immunodeficiency disease (SCID), generally characterized by the absence of mature B and T lymphocytes, but presence of natural killer (NK) cells. Biochemically, mutations in the RAG genes result either in nonfunctional proteins or in proteins with partial recombination activity" (Noordzij, 2002).

The most notable disease resulting from mutations in the RAG-1/RAG-2 proteins is Omenn Syndrome. Omenn syndrome is an autosomal recessive form of severe combined immunodeficiency (SCID) characterized by erythroderma (intense wide spread reddening of the skin), desquamation (the shedding of skin), chronic diarrhea, failure to thrive, lymphadenopathy (swelling of the lymph nodes), and hepatosplenomegaly (abnormal enlargement of both the liver and spleen) (Santagata, 2000). In 2000, Wada et al. found Omenn Syndrome to be attributed to missence mutations in the RAG-1 or RAG-2 gene resulting in partial or no V(D)J recombination activity (Wada, 2000). This mutation leading to the inability of the cell to undergo V(D)J recombination results in non-functional or poorly functional B and T cells (Shigeoka, 2002). This disease has been found in most countries of the world, but mainly North America and Europe. Omenn Syndrome occurs in 1:50,000 individuals in the United States (Shigeoka, 2002). Patients often suffer from opportunistic fungal, bacterial and viral infections (Shigeoka, 2002)

The most successful hope of treatment is a bone marrow transplant, although life threatening rejection is a contraindication. The patient is routinely isolated to prevent infection and ancillary therapy includes intravenous immunoglobulin (IVIG) replacement. Live viral vaccines are also administered (Shigeoka, 2002).


With further developments in gene therapy technology the identification of the recombinase mutations as the cause of Omenn syndrome should enable gene transfer therapy. At this time, successful gene therapy is available only for the X-linked form of SCID, in which mutations in the common g chain are necessary for function of the cell surface receptors of IL-2, IL-4, interleukin 7 (IL-7), interleukin 9 (IL-9), and interleukin 15 (IL-15) (Shigeoka). These breakthroughs however hold great promise for the possible treatment of Omenn syndrome.

Visit Dr. Shigeoka's article for pictures of infants with Omenn Syndrome. (Pictures are located at the bottom of the page)


I was unable to find any evidence in my literature search of drugs binding specifically to RAG-1 or RAG-2.


Ban, C., Ramakrishnan, B., Sundaralingam, M.: "Crystal structure of the highly distorted chimeric decamer r(C)d(CGGCGCCG)r(G).spermine complex--spermine binding to phosphate only and minor groove tertiary base-pairing." Nucleic Acids Res 22: 5466 (1994)
[ Medline ]

Bassing CH, Swat W, Alt FW. (2002) "The mechanism and regulation of chromosomal V(D)J recombination." Cell 109: S45-S55.

Goodsell, D. S., Kopka, M. L., Dickerson, R. E. "Refinement of netropsin bound to DNA: bias and feedback in electron density map interpretation." Biochemistry 34: 4983 (1995)
[ Medline ]

Janeway, C.A., Travers, P., Walport, M., Schlomchik, M. Immunobiology 5: The Immune System in Health and Disease. New York: Garland Publishing, 2001.

Noordzij JG, de Bruin-Versteeg S, Verkaik NS, Vossen JM, de Groot R, Bernatowska E, Langerak AW, van Gent DC, van Dongen JJ.(2002) "The immunophenotypic and immunogenotypic B-cell differentiation arrest in bone marrow of RAG-deficient SCID patients corresponds to residual recombination activities of mutated RAG proteins." Blood 100: 2145-52.

Santagata, S., Gomez, C., Sobacchi, C., Bozzi, F., Abinun, M., Pasic, S., Cortes, P., Vezzoni, P., Villa, A. (2000) "N-Terminal RAG1 Frameshift Mutations in Omenn's Syndrome: INternal Methionine Usage Leads to Partial V(D)J Recombination Activity and Reveals A Fundamental Role in vivo for the N-terminal Domains." Proc. National Academy of Science USA 97: 14572-14577.

Shigeoka, A.O. (2002) "Omenn Syndrome." Inc. (Accessed 3/21/03).

Spanopoulou, E., Zaitseva, F., Wang, F.-H., Santagatoa, S., Baltimore, D., and Panayotou, G. (1996) "The homeodomain region of RAG-1 reveals the parallel mechanisms of bacterial and V(D)J recombination." Cell 87: 263-276.

Wenhui, L., Chang, F.C., and Desiderio, S. (2001) "RAG-1 Mutations with B-Cell-Negative SSCID Dissociate the Nicking and Transesterification Steps of V(D)J Recombination." Molecular and Cellular Biology 21: 3935-3946.

Wada, T., Takei, K., Kudo, M., Shimura, S., Kasahara, Y., Koizumi, S., Kawa-Ha, K., Ishida, Y., Imashuku, S., Seki, H., Yachie, A. (2000) "Characterization of Immune Function and Analysis of RAG Gene Mutations in OMenn Syndrome and Related Disorders." Clinical Exp Immunology 119: 148-155.

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